The overall function of the Cell and Molecular Biology (CMB) Core will be to provide the program with carefully controlled and characterized human cell cultures that are needed in each project. Services provided by the core wjll include the isolation, culture, storage and distribution of primary melanocytes, keratinocytes and fibroblasts. In addition, the CMB Core will also culture and distribute all of the melanoma cell lines that will also be characterized for DNA mutations in B-RAF, N-RAS and PTEN. The CMB Core will ensure that all cells used in the program are free of contamination with mycoplasma or other contaminants by providing regular testing and decontamination when necessary.The CMB core will generate virus for transduction of primary melanocytes with melanomagenic genes or shRNA's to knockdown DNAdamage response genes. Transduced cells will be characterized for expression and function of these genes and knockdown vectors using informative assays such as western blot analysis. The CMB Core will generate in vitro human skin reconstructs (also referred to as orgariotypic skin cultures or skin equivalents) in which artificial human skin is created in vitro in a three-dimensional system with a mixture of primary melanocytes, keratinocytes and fibroblasts. These skin reconstructs will be supplied to Project 1 for in vitro analysis of DNA damage response and Project 3 for engraftment on the backs of SCID mice for in vivo analysis of DNA damage response. The PI of the CMB Core (Dr. Shields) recently completed an NCI-sponsored two-week training program at the Wistar Institute with Dr. Meenhard Herlyn (an internationally recognized leader in melanoma research and Program externaladvisor) to gain hands-on training in preparation of human skin reconstructs. The Cell and Molecular Biology core serves the Program Project by providing important biological reagents to program investigators.